Gherlin analogs

ABSTRACT

The present invention features truncated ghrelin analogs active at the GHS receptor. Ghrelin is a naturally occurring modified peptide. The analogs can bind to the GHS receptor and, preferably, bring about signal transduction. Ghrelin analogs have a variety of different uses including being used as a research tool and being used therapeutically.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to provisionalapplication U.S. Serial No. 60/207,920, filed May 30, 2000, herebyincorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] The references cited herein are not admitted to be prior art tothe claimed invention.

[0003] The pulsatile release of growth hormone from the pituitarysomatotrops is regulated by two hypothalamic neuropeptides: growthhormone-releasing hormone and somatostatin. Growth hormone-releasinghormone stimulates release of growth hormone, whereas, somatostatininhibits secretion of growth hormone. (Frohman et al., Endocronol. Rev.1986, 7, 223-253, and Strobl et al., Pharmacol. Rev. 1994, 46, 1-34.)

[0004] Release of growth hormone from the pituitary somatotrops can alsobe controlled by growth hormone-releasing peptides. A hexapeptideHis-D-Trp-Ala-Trp-D-Phe-Lys-amide (GHRP-6), was found to release growthhormone from somatotrops in a dose-dependent manner in several speciesincluding man. (Bowers et al., Endocrinology 1984, 114, 1537-1545.)Subsequent chemical studies on GHRP-6 led to the identification of otherpotent growth-hormone secretagogues such as GHRP-1, GHRP-2 and hexarelin(Cheng et al., Endocrinology 1989, 124, 2791-2798, Bowers, C. Y. NovelGH-Releasing Peptides. In: Molecular and Clinical Advances in PituitaryDisorders. Ed: Melmed, S.; Endocrine Research and Education, Inc., LosAngeles, Calif., USA 1993, 153-157, and Deghenghi et al., Life Sci.1994, 54, 1321-1328): GHRP-1 Ala-His-D-(2′)-Nal-Ala-Trp-D-Phe-Lys-NH₂GHRP-2 D-Ala-D-(2′)-Nal-Ala-Trp-D-Nal-Lys-NH₂ HexarelinHis-D-2-MeTrp-Ala-Trp-D-Phe-Lys-NH₂.

[0005] GHRP-1, GHRP-2, GHRP-6, and hexarelin are syntheticgrowth-hormone secretagogues. Growth-hormone secretagogues stimulatesecretion of growth hormone by a mechanism different from that of growthhormone-releasing hormone, but like growth hormone-releasing hormone,they antagonize release of somatostatin from the pituitary andhypothalamus. (Bowers et al., Endocrinology 1984, 114, 1537-1545, Chenget al., Endocrinology 1989, 124, 2791-2798, Bowers, C. Y. NovelGH-Releasing Peptides. In: Molecular and Clinical Advances in PituitaryDisorders. Ed: Melmed, S.; Endocrine Research and Education, Inc., LosAngeles, Calif., USA 1993, 153-157, and Deghenghi et al., Life Sci.1994, 54, 1321-1328.)

[0006] The low oral bioavailability (<1%) of the peptidyl growth-hormonesecretagogues stimulated search for non-peptide compounds mimickingaction of GHRP-6 in the pituitary. Several benzolactams and spiroindaneshave been reported to stimulate growth-hormone release in various animalspecies and in man. (Smith et al., Science 1993, 260, 1640-1643,Patchett et al., Proc. Natl. Acad. Sci. USA. 1995, 92, 7001-7005, andChen et al., Bioorg. Med. Chem. Lett. 1996, 6, 2163-2169.) A specificexample of a small spiroindane is MK-0677 (Patchett et al. Proc. Natl.Acad. Sci. USA. 1995, 92, 7001-7005):

[0007] The actions of the above-mentioned growth-hormone secretagogues(both peptide and non-peptide) appear to be mediated by a specificgrowth-hormone secretagogue receptor (GHS receptor). (Howard et al.,Science 1996, 273, 974-977, and Pong et al., Molecular Endocrinology1996, 10, 57-61.) This receptor is present in the pituitary andhypothalamus of various mammalian species (GHSR1a) and is distinct fromthe growth hormone-releasing hormone receptor. The GHS receptor was alsodetected in the other areas of the central nervous system and inperipheral tissues, for instance adrenal and thyroid glands, heart,lung, kidney and skeletal muscles. (Chen et al., Bioorg. Med. Chem.Lett. 1996, 6, 2163-2169, Howard et al., Science 1996, 273, 974-977,Pong et al., Molecular Endocrinology 1996, 10, 57-61, Guan et al., Mol.Brain Res. 1997, 48, 23-29, and McKee et al., Genomics 1997, 46,426-434.) A truncated version of GHSR1a has been reported. (Howard etal., Science 1996, 273, 974-977.)

[0008] The GHS receptor is a G-protein coupled-receptor. Effects of GHSreceptor activation includes depolarization and inhibition of potassiumchannels, an increase in intercellular concentrations of inositoltriphosphate (IP3), and a transient increase in the concentrations ofintracellular calcium. (Pong et al., Molecular Endocrinology 1996, 10,57-61, Guan et al., Mol. Brain Res. 1997, 48, 23-29, and McKee et al.,Genomics 1997, 46, 426-434.)

SUMMARY OF THE INVENTION

[0009] The present invention features truncated ghrelin analogs activeat the GHS receptor. Ghrelin is a naturally occurring modified peptide.The analogs can bind to the GHS receptor and, preferably, bring aboutsignal transduction. Ghrelin analogs have a variety of different usesincluding being used as a research tool and being used therapeutically.

[0010] The structure of human ghrelin is as follows (where the chemicalstructure of a modified serine is shown, and unmodified amino acids arenoted using one letter codes):

[0011] A core region present in ghrelin was found to provide foractivity at the GHS receptor. The core region comprises the fourN-terminal amino acids, where the serine at position 2 or 3 is modifiedwith a bulky hydrophobic R group.

[0012] Thus, a first aspect of the present invention describes atruncated ghrelin analog having a structure selected from the groupconsisting of:

[0013] a) Z¹-GSXF(Z)_(n)—Z²; and

[0014] b) Z¹-GXSF(Z)_(n)—Z²;

[0015] wherein X is a modified amino acid containing a bulky hydrophobicR group;

[0016] each Z is independently either alanine, valine, leucine,isoleucine, proline, tryptophan, phenylalanine, methionine, glycine,serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,arginine, histidine, aspartic acid, or glutamic acid, or a derivativethereof;

[0017] Z¹ is an optionally present protecting group that, if present, iscovalently joined to the N-terminal amino group;

[0018] Z² is an optionally present protecting group that, if present, iscovalently joined to the C-terminal carboxy group; and

[0019] n is 0 to 19;

[0020] or a pharmaceutically acceptable salt thereof.

[0021] Unless otherwise stated, those amino acids with a chiral centerare provided in the L-enantiomer. Reference to “a derivative thereof”refers to a modified amino acid such as the corresponding D-amino acid,a N-alkyl-amino acid, a β-amino acid, or a labeled amino acid.

[0022] Another aspect of the present invention describes a method ofscreening for a compound able to bind to a GHS receptor. The methodcomprises the step of measuring the ability of a compound to affectbinding of a truncated ghrelin analog to either the receptor, a fragmentof the receptor comprising a ghrelin binding site, a polypeptidecomprising the fragment, or a derivative of the polypeptide.

[0023] Another aspect of the present invention describes a method forachieving a beneficial affect in a subject comprising the step ofadministering to the subject an effective amount of a ghrelin analog.The effective amount produces a beneficial effect in helping to treat(e.g., cure or reduce the severity) or prevent (e.g., reduce thelikelihood of onset or severity) a disease or disorder.

[0024] Another aspect of the present invention describes a method forstimulating growth hormone secretion comprising the step ofadministering to a subject an effective amount of a ghrelin analog. Theeffective amount is at least an amount sufficient to produce adetectable increase in growth hormone secretion and, preferably, is anamount sufficient to achieve a beneficial affect in a patient.

[0025] Other features and advantages of the present invention areapparent from the additional descriptions provided herein including thedifferent examples. The provided examples illustrate differentcomponents and methodology useful in practicing the present invention.The examples do not limit the claimed invention. Based on the presentdisclosure the skilled artisan can identify and employ other componentsand methodology useful for practicing the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention features truncated ghrelin analogs activeat the GHS receptor. Human ghrelin is a 28 amino acid modified peptidewhere a serine hydroxyl group is esterified by n-octanoic acid. (Kojimaet al., Nature 1999, 402, 656-660, and Kojima, (Abstract), ThirdInternational Symposium on Growth Hormone Secretagogues, Keystone,Colo., USA Feb. 17-19, 2000.)

[0027] Ghrelin induces growth hormone release from primary-culturepituitary cells in a dose-dependent manner without stimulating therelease of the other pituitary hormones. Injected intravenously intoanaesthetized rats, ghrelin stimulated pulsatile release of growthhormone. (Kojima et al., Nature 1999, 402, 656-660.)

[0028] Truncated ghrelin analogs described herein are active at the GHSreceptor. The analogs can bind to the receptor, and preferably,stimulate receptor activity. Ghrelin analogs have a variety of differentuses including being used as a research tool and being usedtherapeutically.

[0029] Research tool applications generally involve the use of atruncated ghrelin analog and the presence of a GHS receptor or fragmentthereof. The GHS receptor can be present in different environments suchas a mammalian subject, a whole cell, or membrane fragments. Examples ofresearch tool applications include screening for compounds active at theGHS receptor, determining the presence of the GHS receptor in a sampleor preparation, and examining the role or effect of ghrelin.

[0030] Ghrelin analogs can be used to screen for both ghrelin agonistsand ghrelin antagonists. Screening for ghrelin agonists can beperformed, for example, by using a ghrelin analog in a competitionexperiment with test compounds. Screening for ghrelin antagonists can beperformed, for example, by using a ghrelin analog to produce GHSreceptor activity and then measuring the ability of a compound to alterGHS receptor activity.

[0031] Ghrelin analogs can be administered to a subject. A “subject”refers to a mammal including, for example, a human, a rat, a mouse, or afarm animal. Reference to subject does not necessarily indicate thepresence of a disease or disorder. The term subject includes, forexample, mammals being dosed with a truncated ghrelin analog as part ofan experiment, mammals being treated to help alleviate a disease ordisorder, and mammals being treated prophylactically to retard orprevent the onset of a disease or disorder.

[0032] Ghrelin agonists can be used to achieve a beneficial effect in asubject such as one or more of the following: treating a growth hormonedeficient state, increasing muscle mass, increasing bone density,treating sexual dysfunction in males or females, facilitating a weightgain, facilitating maintenance of weight, facilitating maintenance ofphysical functioning, facilitating recovery of physical function, and/orfacilitating appetite increase. Facilitating a weight gain, maintenancein weight, or appetite increase is particularly useful for a patienthaving a disease or disorder, or under going a treatment, accompanied byweight loss. Examples of diseases or disorders accompanied by weightloss include anorexia, bulimia, cancer cachexia, AIDS, wasting,cachexia, and wasting in frail elderly. Examples of treatmentsaccompanied by weight loss include chemotherapy, radiation therapy,temporary or permanent immobilization, and dialysis.

[0033] Ghrelin antagonists can also be used to achieve a beneficialeffect in a patient. For example, a ghrelin antagonist can be used tofacilitate weight loss, facilitate appetite decrease, facilitate weightmaintenance, treat obesity, treat diabetes, treat complications ofdiabetes including retinopathy, and/or treat cardiovascular disorders.Excessive weight is a contributing factor to different diseasesincluding hypertension, diabetes, dyslipidemias, cardiovascular disease,gall stones, osteoarthritis and certain forms of cancers. Bringing abouta weight loss can be used, for example, to reduce the likelihood of suchdiseases and as part of a treatment for such diseases.

[0034] Truncated Ghrelin Analogs

[0035] The smaller size of truncated ghrelin analogs offers advantagesover longer-length ghrelin such as ease of synthesis and/or increasedsolubility in physiological buffers. In addition, small analogs canserve as models for producing peptidomimetics having desirablepharmacological properties.

[0036] Truncated ghrelin analogs described herein have the structure:

Z¹-GSXF(Z)_(n)—Z² or Z¹-GXSF(Z)_(n)—Z²; preferably, Z¹-GSXF(Z)_(n)—Z².

[0037] wherein X is a modified amino acid containing a bulky hydrophobicR group;

[0038] each Z is independently either alanine, valine, leucine,isoleucine, proline, tryptophan, phenylalanine, methionine, glycine,serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,arginine, histidine, aspartic acid, or glutamic acid, or a derivativethereof;

[0039] Z¹ is an optionally present protecting group that, if present, iscovalently joined to the N-terminal amino group;

[0040] Z² is an optionally present protecting group that, if present, iscovalently joined to the C-terminal carboxy group; and

[0041] n is 0 to 19; in different embodiments n is 0-3, 0-6, 0-11, 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or11;

[0042] or a pharmaceutically acceptable salt thereof.

[0043] Individual amino acids such as those present in the generalstructure of a truncated ghrelin analog or a (Z)_(n) group can berepresented as follows: A=Ala=Alanine; C=Cys=Cysteine; D=Asp=Asparticacid; E=Glu=Glutamic acid; F=Phe=Phenylalanine; G=Gly=Glycine;H=His=Histidine; I=Ile=Isoleucine; K=Lys=Lysine; L=Leu=Leucine;M=Met=Methionine; N=Asn=Asparagine; P=Pro=Proline; Q=Gln=Glutamine;R=Arg=Arginine; S=Ser=Serine; T=Thr=Threonine; V=Val=Valine;W=Trp=Tryptophan; and Y=Tyr=Tyrosine.

[0044] The present invention includes diastereomers as well as theirracemic and resolved enantiomerically pure forms. Truncated ghrelinanalogs can contain D-amino acids, L-amino acids or a combinationthereof. Preferably, amino acids present in a truncated ghrelin analogare the L-enantiomer.

[0045] Since each Z is independently selected, the resulting (Z)_(n)group is not limited to a group of contiguous amino acids present inghrelin. In an embodiment of the present invention (Z)_(n) is acontiguous amino acid group present in ghrelin.

[0046] The design of analogs where (Z)_(n) is a contiguous amino acidgroup not present in ghrelin can be based on substitutions to ghrelin,or a truncated ghrelin, where the substitutions take into accountdifferences in amino acids R groups. An R group affects differentproperties of the amino acid such as physical size, charge, andhydrophobicity. Amino acids can be divided into different groups asfollows: neutral and hydrophobic (alanine, valine, leucine, isoleucine,proline, tryptophan, phenylalaine, and methionine); neutral and polar(glycine, serine, threonine, tyrosine, cysteine, asparagine, andglutamine); basic (lysine, arginine, and histidine); and acidic(aspartic acid and glutamic acid).

[0047] Generally, in substituting different amino acids it is preferableto exchange amino acids having similar properties. Substitutingdifferent amino acids within a particular group, such as substitutingvaline for leucine, arginine for lysine, and asparagine for glutamineare good candidates for not causing a change in peptide functioning.

[0048] Preferred derivatives are D-amino acids, N-allyl-amino acids,β-amino acids, or a labeled amino acid (including a labeled version of aD-amino acid, a N-alkyl-amino acids, or a β-amino acid). A labeledderivative indicates the alteration of an amino acid or amino acidderivative with a detectable label. Examples of detectable labelsinclude luminescent, enzymatic, and radioactive labels. Both the type oflabel and the position of the label can effect analog activity. Labelsshould be selected and positioned so as not to substantially alter theactivity of the truncated ghrelin analog at the GHS receptor. The effectof a particular label and position on ghrelin activity can be determinedusing assays measuring ghrelin activity and/or binding.

[0049] A protecting group covalently joined to the N-terminal aminogroup reduces the reactivity of the amino terminus under in vivoconditions. Amino protecting groups include —C₁₋₁₀ alkyl, —C₁₋₁₀substituted alkyl, —C₂₋₁₀ alkenyl, —C₂₋₁₀ substituted alkenyl, aryl,—C₁₋₆ alkyl aryl, —C(O)—(CH₂)₁₋₆—COOH, —C(O)—C₁₋₆ alkyl, —C(O)-aryl,—C(O)—O—C₁₋₆ alkyl, or —C(O)—O-aryl. Preferably, the amino terminusprotecting group is acetyl, propyl, succinyl, benzyl, benzyloxycarbonylor t-butyloxycarbonyl.

[0050] A protecting group covalently joined to the C-terminal carboxygroup reduces the reactivity of the carboxy terminus under in vivoconditions. The carboxy terminus protecting group is preferably attachedto the α-carbonyl group of the last amino acid. Carboxy terminusprotecting groups include amide, methylamide, and ethylamide.

[0051] In a preferred embodiment X has the structure:

[0052] wherein X¹ is either —O—, —S—, —OC(O)—, —NHC(O)—, or —CH₂—;preferably, X¹ is either —OC(O)— or —NHC(O)—, more preferably, X¹ is—OC(O)—; and

[0053] R is either a longer length alkyl, substituted longer lengthalkyl, longer length alkenyl, substituted longer length alkenyl, longerlength heteroalkyl, substituted longer length heteroalkyl, aryl, oralkylaryl; preferably, R is either —C₄₋₂₀ alkyl, —C₄₋₂₀ substitutedalkyl, —C₄₋₂₀ substituted alkenyl, —C₄₋₂₀ alkenyl, —C₄₋₂₀ heteroalkyl,—C₄₋₂₀ substituted heteroalkyl, aryl, or alkylaryl; more preferably, Ris a —C₅₋₁₅ alkyl or a —C₅₋₁₅ substituted alkyl; more preferably R is a—C₅₋₁₅; more preferably, R is —(CH₂)₆CH₃.

[0054] Examples of truncated ghrelin analogs including the followingmodified peptides:

[0055] GSXFLSPEHQRVQQ (compound 13, SEQ. ID. NO. 2),

[0056] GSX^(b)FLSPEHQRVQQ (compound 14, SEQ. ID. NO. 3),

[0057] GSXFLSPEHQRVQQRKESKKPPA-NH₂ (compound 18, SEQ. ID. NO. 4),

[0058] GSXFLSPEHQRVQQRKES-NH₂ (compound 19, SEQ. ID. NO. 5),

[0059] GSXFLSPEHQ-NH₂ (compound 20, SEQ. ID. NO. 6),

[0060] GSXFL-NH₂ (compound 21),

[0061] GSXF-NH₂ (compound 22),

[0062] wherein X is

[0063] and X^(b) is

[0064] The effect of particular groups, such as X, (Z)_(n) or blockinggroups on ghrelin analog activity at the GHS receptor can be determinedusing techniques such as those described in the examples provided below.In different embodiments a truncated ghrelin analog has at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90%, functional activity relative to ghrelin as determinedusing the assay described in the Example 3 infra; and/or has an IC₅₀greater than about 1,000, greater than about 100, or greater than about50 using the binding assay described in Example 2 infra. With respect toIC₅₀, greater refers to potency and thus indicates a lesser amount isneeded to achieve binding inhibition.

[0065] Truncated ghrelin analogs can be produced using techniques wellknown in the art. For example, a polypeptide region of a truncatedghrelin analog can be chemically or biochemically synthesized andmodified. Techniques for chemical synthesis of polypeptides are wellknown in the art. (See e.g., Vincent in Peptide and Protein DrugDelivery, New York, N.Y., Dekker, 1990.) Examples of techniques forbiochemical synthesis involving the introduction of a nucleic acid intoa cell and expression of nucleic acids are provided in Ausubel, CurrentProtocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook etal., in Molecular Cloning, A Laboratory Manual, 2^(nd) Edition, ColdSpring Harbor Laboratory Press, 1989.

[0066] Chemical Definitions

[0067] Definitions of some of the chemical groups making up a protectinggroup or present in an R group are provided below.

[0068] “Alkyl” refers to a hydrocarbon group containing one or morecarbon atoms, where multiple carbon atoms if present are joined bysingle bonds. The alkyl hydrocarbon group may be straight-chain orcontain one or more branches or cyclic groups. Protecting grouphydrocarbons are preferably C₁₋₄. R alkyl groups are preferably about 4to about 20 carbon atoms in length, more preferably C₅₋₁₅, and morepreferably —(CH₂)₆CH₃. Reference to “longer length alkyl” indicates atleast about 4 carbon atoms.

[0069] “Substituted alkyl” refers to an alkyl wherein one or morehydrogens of the hydrocarbon group are replaced with one or moresubstituents selected from the group consisting of halogen (preferably—F or —Cl), —OH, —CN, —SH, —NH₂, —NHCH₃, —NO₂, —C₁₋₂ alkyl substitutedwith 1 to 6 halogens (preferably —F or —Cl, more preferably —F), —CF₃,—OCH₃, —OCF₃, and —(CH₂)₀₋₄—COOH. In different embodiments 1, 2, 3 or 4substituents are present. The presence of —(CH₂)₀₋₄—COOH results in theproduction of an alkyl acid. Examples of alkyl acids containing, orconsisting of, —(CH₂)₀₋₄—COOH include 2-norbornane acetic acid,tert-butyric acid and 3-cyclopentyl propionic acid.

[0070] “Heteroalkyl” refers to an alkyl wherein one of more of thecarbon atoms in the hydrocarbon group are replaced with one or more ofthe following groups: amino, amido, —O—, or carbonyl. In differentembodiments 1 or 2 heteroatoms are present.

[0071] “Substituted hetereoalkyl” refers to a heteroalkyl wherein one ormore hydrogens of the hydrocarbon group are replaced with one or moresubstituents selected from the group consisting of halogen (preferably—F or —Cl), —OH, —CN, —SH, —NH₂, —NHCH₃, —NO₂, —C₁₋₂ alkyl substitutedwith 1 to 6 halogens (preferably —F or —Cl, more preferably —F), —CF₃,—OCH₃, —OCF₃, and —(CH₂)₀₋₄—COOH. In different embodiments 1, 2, 3 or 4substituents are present.

[0072] “Alkenyl” refers to a hydrocarbon group made up of two or morecarbons where one or more carbon-carbon double bonds are present. Thealkenyl hydrocarbon group may be straight-chain or contain one or morebranches or cyclic groups. Protecting group hydrocarbons are preferablyC₂₋₄. R alkenyl groups are preferably about 4 to about 20 carbon atomsin length, more preferably C₅₋₁₅. Reference to “longer length alkenyl”indicates at least about 4 carbon atoms.

[0073] “Substituted alkenyl” refers to an alkenyl wherein one or morehydrogens are replaced with one or more substituents selected from thegroup consisting of halogen (preferably —F or —Cl), —OH, —CN, —SH, —NH₂,—NHCH₃, —NO₂, —C₁₋₂ alkyl substituted with 1 to 6 halogens (preferably—F or —Cl, more preferably —F), —CF₃, —OCH₃, —OCF₃, and —(CH₂)₀₋₄—COOH.In different embodiments 1, 2, 3 or 4 substituents are present.

[0074] “Aryl” refers to an optionally substituted aromatic group with atleast one ring having a conjugated pi-electron system, containing up totwo conjugated or fused ring systems. Aryl includes carbocyclic aryl,heterocyclic aryl and biaryl groups. Preferably, the aryl is a 5 or 6membered ring. Preferred atoms for a heterocyclic aryl are one or moresulfur, oxygen, and/or nitrogen. Examples of aryl include phenyl,1-naphthyl, 2-naphthyl, indole, quinoline, 2-imidazole, and 9-antracene.Aryl substituents are selected from the group consisting of —C₁₋₄ alkyl,—C₁₋₄ alkoxy, halogen (preferably —F or —Cl), —OH, —CN, —SH, —NH₂, —NO₂,—C₁₋₂ alkyl substituted with 1 to 5 halogens (preferably —F or —Cl, morepreferably —F), —CF₃, —OCF₃, and —(CH₂)₀₋₄—COOH. In differentembodiments the aryl contains 0, 1, 2, 3, or 4 substituents.

[0075] “Alkylaryl” refers to an “alkyl” joined to an “aryl”.

[0076] GHS Receptor Binding Assay

[0077] Assays measuring the ability of a compound to bind a GHS receptoremploy a GHS receptor, a fragment of the receptor comprising a ghrelinbinding site, a polypeptide comprising such a fragment, or a derivativeof the polypeptide. Preferably, the assay uses the GHS receptor or afragment thereof.

[0078] A polypeptide comprising a GHS receptor fragment that bindsghrelin can also contain one or more polypeptide regions not found in aGHS receptor. A derivative of such a polypeptide comprises a GHSreceptor fragment that binds ghrelin along with one or more non-peptidecomponents.

[0079] The GHS receptor amino acid sequence involved in ghrelin bindingcan be readily identified using labeled ghrelin or truncated ghrelinanalogs and different receptor fragments. Different strategies can beemployed to select fragments to be tested to narrow down the bindingregion. Examples of such strategies include testing consecutivefragments about 15 amino acids in length starting at the N-terminus, andtesting longer length fragments. If longer length fragments are tested,a fragment binding ghrelin can be subdivided to further locate theghrelin binding region. Fragments used for binding studies can begenerated using recombinant nucleic acid techniques.

[0080] Binding assays can be performed using individual compounds orpreparations containing different numbers of compounds. A preparationcontaining different numbers of compounds having the ability to bind tothe GHS receptor can be divided into smaller groups of compounds thatcan be tested to identify the compound(s) binding to the GHS receptor.In an embodiment of the present invention, a test preparation containingat least 10 compounds is used in a binding assay.

[0081] Binding assays can be performed using recombinantly produced GHSreceptor polypeptides present in different environments. Suchenvironments include, for example, cell extracts and purified cellextracts containing the GHS receptor polypeptide expressed fromrecombinant nucleic acid or naturally occurring nucleic acid; and alsoinclude, for example, the use of a purified GHS receptor polypeptideproduced by recombinant means or from naturally occurring nucleic acidwhich is introduced into a different environment.

[0082] Screening for GHS Receptor Active Compounds

[0083] Screening for GHS receptor active compounds is facilitated usinga recombinantly expressed receptor. Using a recombinantly expressed GHSreceptor offers several advantages such as the ability to express thereceptor in a defined cell system so that a response to a compound atthe GHS receptor can more readily be differentiated from responses atother receptors. For example, the GHS receptor can be expressed in acell line such as HEK 293, COS 7, and CHO not normally expressing thereceptor by an expression vector, wherein the same cell line without theexpression vector can act as a control.

[0084] Screening for compounds reducing GHS receptor activity isfacilitated through the use of a truncated ghrelin analog in the assay.The use of a truncated ghrelin analog in a screening assay provides forGHS receptor activity. The effect of test compounds on such activity canbe measured to identify, for example, allosteric modulators andantagonists.

[0085] GHS receptor activity can be measured using different techniquessuch as detecting a change in the intracellular conformation of the GHSreceptor, in the G-protein coupled activities, and/or in theintracellular messengers. Preferably, GHS receptor activity is measuredusing techniques such as those measuring intracellular Ca²⁺. Examples oftechniques well known in the art that can be employed to measure Ca²⁺include the use of dyes such as Fura-2 and the use ofCa²⁺-bioluminescent sensitive reporter proteins such as aequorin. Anexample of a cell line employing aequorin to measure G-protein activityis HEK293/aeq17. (Button et al., 1993, Cell Calcium 14, 663-671, andFeighner et al., 1999, Science 284, 2184-2188.)

[0086] Chimeric receptors containing a ghrelin binding regionfunctionally coupled to a different G-protein can also be used tomeasure GHS receptor activity. A chimeric GHS receptor contains anN-terminal extracellular domain; a transmembrane domain made up oftransmembrane regions, extracellular loop regions, and intracellularloop regions; and an intracellular carboxy terminus. Techniques forproducing chimeric receptors and measuring G-protein coupled responsesare provided in, for example, International Application Number WO97/05252, and U.S. Pat. No. 5,264,565, both of which are herebyincorporated by reference herein.

[0087] Stimulation of GHS Receptor Activity

[0088] Truncated ghrelin analogs can be used to stimulate GHS receptoractivity. Such stimulation can be used, for example, to study the effectof GHS receptor modulation, to study the effect of growth hormonesecretion, to look for or study ghrelin antagonists, or to achieve abeneficial effect in a subject. Beneficial effects that can be achievedinclude one or more of the following: treating a growth hormonedeficient state, increasing muscle mass, increasing bone density,treating sexual dysfunction in males or females, facilitating a weightgain, facilitating maintenance of weight, facilitating maintenance ofphysical functioning, facilitating recovery of physical function, and/orfacilitating appetite increase.

[0089] Increasing weight or appetite can be useful for maintainingweight or producing a weight or appetite gain in an under weightsubject, or in a patient having a disease or undergoing treatment thataffects weight or appetite. In addition, for example, farm animals suchas pigs, cows and chickens can be treated to gain weight.

[0090] Under weight subjects include those having a body weight about10% or less, 20% or less, or 30% or less, than the lower end of a“normal” weight range or Body Mass Index (“BMI”). “Normal” weight rangesare well known in the art and take into account factors such as apatient age, height, and body type.

[0091] BMI measures your height/weight ratio. It is determined bycalculating weight in kilograms divided by the square of height inmeters. The BMI “normal” range is 19-22.

[0092] Administration

[0093] Truncated ghrelin analogs can be formulated and administered to asubject using the guidance provided herein along with techniques wellknown in the art. The preferred route of administration ensures that aneffective amount of compound reaches the target. Guidelines forpharmaceutical administration in general are provided in, for example,Remington's Pharmaceutical Sciences 18^(th) Edition, Ed. Gennaro, MackPublishing, 1990, and Modern Pharmaceutics 2^(nd) Edition, Eds. Bankerand Rhodes, Marcel Dekker, Inc., 1990, both of which are herebyincorporated by reference herein.

[0094] Truncated ghrelin analogs can be prepared as acidic or basicsalts. Pharmaceutically acceptable salts (in the form of water- oroil-soluble or dispersible products) include conventional non-toxicsalts or the quaternary ammonium salts that are formed, e.g., frominorganic or organic acids or bases. Examples of such salts include acidaddition salts such as acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate;and base salts such as ammonium salts, alkali metal salts such as sodiumand potassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such as arginineand lysine.

[0095] Truncated ghrelin analogs can be administered using differentroutes including oral, nasal, by injection, transdermal, andtransmucosally. Active ingredients to be administered orally as asuspension can be prepared according to techniques well known in the artof pharmaceutical formulation and may contain microcrystalline cellulosefor imparting bulk, alginic acid or sodium alginate as a suspendingagent, methylcellulose as a viscosity enhancer, and sweeteners/flavoringagents. As immediate release tablets, these compositions may containmicrocrystalline cellulose, dicalcium phosphate, starch, magnesiumstearate and lactose and/or other excipients, binders, extenders,disintegrants, diluents and lubricants.

[0096] Administered by nasal aerosol or inhalation formulations may beprepared, for example, as solutions in saline, employing benzyl alcoholor other suitable preservatives, absorption promoters to enhancebioavailability, employing fluorocarbons, and/or employing othersolubilizing or dispersing agents.

[0097] Truncated ghrelin analogs may also be administered in intravenous(both bolus and infusion), intraperitoneal, subcutaneous, topical withor without occlusion, or intramuscular form. When administered byinjection, the injectable solution or suspension may be formulated usingsuitable non-toxic, parenterally-acceptable diluents or solvents, suchas Ringer's solution or isotonic sodium chloride solution, or suitabledispersing or wetting and suspending agents, such as sterile, bland,fixed oils, including synthetic mono- or diglycerides, and fatty acids,including oleic acid.

[0098] Suitable dosing regimens are preferably determined taking intoaccount factors well known in the art including type of subject beingdosed; age, weight, sex and medical condition of the subject; the routeof administration; the renal and hepatic function of the subject; thedesired effect; and the particular compound employed.

[0099] Optimal precision in achieving concentrations of drug within therange that yields efficacy without toxicity requires a regimen based onthe kinetics of the drug's availability to target sites. This involves aconsideration of the distribution, equilibrium, and elimination of adrug. The daily dose for a subject is expected to be between 0.01 and1,000 mg per subject per day.

[0100] Truncated ghrelin analogs can be provided in kit. Such a kittypically contains an active compound in dosage forms foradministration. A dosage form contains a sufficient amount of activecompound such that a desirable effect can be obtained when administeredto a subject during regular intervals, such as 1 to 6 times a day,during the course of 1 or more days. Preferably, a kit containsinstructions indicating the use of the dosage form to achieve adesirable affect and the amount of dosage form to be taken over aspecified time period.

EXAMPLES

[0101] Examples are provided below to further illustrate differentfeatures of the present invention. The examples also illustrate usefulmethodology for practicing the invention. These examples do not limitthe claimed invention.

Example 1

[0102] Peptide Synthesis Purification and Characterization

[0103] Fmoc-protected amino acids were obtained from AnaSpec (San Jose,Calif.), 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin fromPE Biosystems (Foster City, Calif.), Boc-7-aminoheptanoic acid fromBachem (King of Prussia, Pa.) and n-octanoic acid, 2,4,6-octatrienoicacid, 2-propylpentanoic acid, 11-undecanoic acid, palmitic acid,8-bromooctanoic acid, 1-adamanteneacetic acid and benzoic acid fromAldrich (Milwaukee, Wis.).

[0104] Elongation of peptidyl chains on4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin was performedon a 431A ABI peptide synthesizer. Manufacture-supplied protocols wereapplied for coupling of the hydroxybenzotriazole esters of amino acidsin N-methylpyrrolidone (NMP). The fluorenylmethyloxycarbonyl (Fmoc)group was used as a semipermanent α-amino protecting group, whereas theside chains protecting groups were: tert-butyl for serine, trityl forserine in position 3 and for histidine and glutamine, tert-butyl esterfor glutamic acid, 2,2,4,6,7,-pentamethyldihydrobenzofuran-5-sulfonyl(Pbf) for arginine, tert-buthyloxycarbonyl (Boc) for lysine and forα-amino group of glycine in position 1.

[0105] The peptidyl resin was then transferred into a vessel and thetrityl group from the side chain of Ser³ was manually removed with 1%trifluoroacetic acid (TFA) in dichloromethane (45 minutes at roomtemperature). The peptidyl resin was thoroughly washed, and thenagitated for 4 hours with the 6-fold excess of each1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) and a selected acidin DCM or NMP, in the presence of a catalytic amount of4-dimethylaminopyridine (ca. 10 mg). The peptidyl resin was again washedwith DCM, NMP and methanol, dried and treated with TFA in the presenceof scavengers (ca. 3% total of the mixture ofwater-anisole-triethylsilane, 1:1:1, v/v/v). After 1.5 hours, the resinwas filtered off, TFA was removed in vacuo and the residue wastriturated with ether. The precipitate which formed was filtered off,washed thoroughly with ether and dried.

[0106] The crude peptide was analyzed by analytical reverse-phasehigh-pressure liquid chromatography (RP HPLC) on a C18 Vydac columnattached to a Waters 600E system with automatic Wisp 712 injector and991 Photodiode Array detector. A standard gradient system of 0-100%buffer B in 30 minutes (G1), or a gradient of 20-80% buffer B in 30minutes (G2), was used for analysis; buffer A was 0.1% trifluoroaceticacid in water and buffer B was 0.1% trifluoroacetic acid inacetonitrile. HPLC profiles were recorded at 210 nm and 230 nm.

[0107] Preparative separations were performed on a Waters Delta Prep4000 system with a semipreparative C18 RP Vydac column. Theabove-described solvent system of water and acetonitrile, in a gradientof 20-80% buffer B in 60 minutes (G3) or in a gradient of 0-60% bufferB, was used for separations.

[0108] The chromatographically homogenous compounds were analyzed byelectrospray mass spectrometry (Hewlett Packard Series 1100 MSDspectrometer).

Example 2

[0109] Binding Assay

[0110] Binding of [³⁵S]-MK-677 to crude membranes prepared from HEK293-aequorin stable cell lines was performed as described in Chen etal., Bioorg. Med. Chem. Lett. 1996, 6, 2163-2169, and Howard et al.,Science 1996, 273, 974-977. Prior to the binding assay, the HEK293-AEQ17 cells (8×10⁵ cells plated 18 hours before transfection in aT75 flask) were transfected with 22 μg of human GHS receptor plasmidusing 264 μg lipofectamine. The open reading frame cDNA (SEQ. ID. NO.22) encoding the human GHS receptor inserted in the mammalian expressionvector pcDNA-3 (Invitrogen, Carlsbad, Calif.) was used for binding andexpression studies.

[0111] For a 96-well filter binding assay, 0.05 nM [³⁵S]-NM-677(specific activity ˜1200 Ci/mmol) was bound to 4 μg membrane protein perwell with or without competing test ligand. The bound membranes werefiltered on 0.5% polyethylenimine prewet filters (UniFilter 96 GF/C;Packard #6005174, Meriden, Conn.). Filters were washed 8 times, dried,and counted with Microscint 20 (Packard #6013621, Meriden, Conn.). IC₅₀values were determined from three separate assays performed intriplicate. SEQ. ID. NO. 22 is as follows:ATGTGGAACGCGACGCCCAGCGAAGAGCCGGGGTTCAACCTCACACTGGCCGACCTGGACTGGGATGCTTCCCCCGGCAACGACTCGCTGGGCGACGAGCTGCTGCAGCTCTTCCCCGCGCCGCTGCTGGCGGGCGTCACAGCCACCTGCGTGGCACTCTTCGTGGTGGGTATCGCTGGCAACCTGCTCACCATGCTGGTGGTGTCGCGCTTCCGCGAGCTGCGCACCACCACCAACCTCTACCTGTCCAGCATGGCCTTCTCCGATCTGCTCATCTTCCTCTGCATGCCCCTGGACCTCGTTCGCCTCTGGCAGTACCGGCCCTGGAACTTCGGCGACCTCCTCTGCAAACTCTTCCAATTCGTCAGTGAGAGCTGCACCTACGCCACGGTGCTCACCATCACAGCGCTGAGCGTCGAGCGCTACTTCGCCATCTGCTTCCCACTCCGGGCCAAGGTGGTGGTCACCAAGGGGCGGGTGAAGCTGGTCATCTTCGTCATCTGGGCCGTGGCCTTCTGCAGCGCCGGGCCCATCTTCGTGCTAGTCGGGGTGGAGCACGAGAACGGCACCGACCCTTGGGACACCAACGAGTGCCGCCCCACCGAGTTTGCGGTGCGCTCTGGACTGCTCACGGTCATGGTGTGGGTGTCCAGCATCTTCTTCTTCCTTCCTGTCTTCTGTCTCACGGTCCTCTACAGTCTCATCGGCAGGAAGCTGTGGCGGAGGAGGCGCGGCGATGCTGTCGTGGGTGCCTCGCTCAGGGACCAGAACCACAAGCAAACCGTGAAAATGCTGGCTGTAGTGGTGTTTGCCTTCATCCTCTGCTGGCTCCCCTTCCACGTAGGGCGATATTTATTTTCCAAATCCTTTGAGCCTGGCTCCTTGGAGATTGCTCAGATCAGCCAGTACTGCAACCTCGTGTCCTTTGTCCTCTTCTACCTCAGTGCTGCCATCAACCCCATTCTGTACAACATCATGTCCAAGAAGTACCGGGTGGCAGTGTTCAGACTTCTGGGATTCGAACCCTTCTCCCAGAGAAAGCTCTCCACTCTGAAAGATGAAAGTTCTCGGGCCTGGACAGAATCTAGTATTAATACATG A

Example 3

[0112] Aeguorin Bioluminescence Functional Assay

[0113] The aequorin bioluminescence assay is a reliable test foridentifying G-protein coupled receptors which couple through the Gαprotein subunit family consisting of Gq and G11. Coupling through Gq andG11 leads to the activation of phospholipase C, mobilization ofintracellular calcium and activation of protein kinase C. A stable cellline expressing the human GHSR1a and the aequorin reporter protein wereused for the assay. (Button et al., Cell Calcium 1993, 14, 663-671.)

[0114] The assay was performed using a Luminoskan RT luminometer(Labsystems Inc., Gaithersburg, Md.) controlled by custom softwarewritten for a Macintosh PowerPC 6100. 293AEQ17/GHSR1a cells (asdescribed in Example 2), were cultured for 72 hours and the apo-aequorinin the cells was charged for 1 hour with coelenterazine (10 μM) underreducing conditions (300 μM reduced glutathione) in ECB buffer (140 mMNaCl, 20 mM KCl, 20 mM HEPES-NaOH [pH=7.4], 5 mM glucose, 1 mM MgCl₂, 1mM CaCl₂, 0.1 mg/ml bovine serum albumin). The cells were harvested,washed once in ECB medium and resuspended to 500,000 cells/ml. 100 ml ofcell suspension (corresponding to 5×10⁴ cells) was then injected intothe test plate containing the peptides, and the integrated lightemission was recorded over 30 seconds, in 0.5 second units. 20 μL oflysis buffer (0.1% final Triton X-100 concentration) was then injectedand the integrated light emission recorded over 10 seconds, in 0.5second units. The “fractional response” values for each well werecalculated by taking the ratio of the integrated response to the initialchallenge to the total integrated luminescence including the TritonX-100 lysis response. The functional EC₅₀ values were measured in threeseparate assays.

Example 4

[0115] Activity of Different Types of Ghlerin Analogs

[0116] Analogs of human ghrelin (Tables 1-4) were prepared by solidphase syntheses as described in Example 1. The analogs were evaluatedfor their binding affinities to the cloned human GHS receptor 1a in acompetitive binding assay with [³⁵S]-MK-677 as the radiolabeled ligand,and also for their ability to stimulate inositol triphosphate-coupledmobilization of intracellular calcium in HEK-293 cells expressinghGHSR1a, as described in Examples 2 and 3.

[0117] The role of the n-octanoyl group in interaction of human ghrelinwith hGHSR1a was examined by testing compounds in which the hydroxylgroup in the side chain of Ser³ was acylated by various aliphatic oraromatic acids (see Table 1). Acylation of Ser³ with hydrophobic acidsresembling in size n-octanoic acid, such as the unsaturated2,4,6-octatrienoic acid or the branched 2-propylpentanoic acid, or thelonger chain 11-undecanoic acid. or palmitic acid, yielded compounds 1-4having agonist potencies similar to that of ghrelin. In contrast,replacement of the n-octanoyl group with the substantially smalleracetyl group led to compound 5 which was 20-fold less potent than humanghrelin in the hGHSR1a activation assay. Ghrelin without the n-octanoylgroup (des-octanoyl-ghrelin) poorly activated hGHSR1a even at micromolarconcentrations (compound 6 in Table 1).

[0118] To explore possible non-hydrophobic interactions with thereceptor, the hydroxyl group of serine 3 in compounds 7, 8, 9 wasacylated with aliphatic acids which mimic the extended hydrophobic chainof n-octanoic acid, but also contain bromo, amino or amido groups. Inthe hGHSR1a activation assay, analogs with amido and amino groups in theside chain of residue 3 were respectively 5- and 20-fold less potent asagonists at hGHSR1a (compounds 8 and 9 in Table 1), whereas, compound 7with 8-bromooctanoyl group attached to the side chain of Ser³ retainedthe potency of the parent compound. Compound 10 having the bulky andrigid hydrophobic 1-adamantaneacetyl group in place of n-octanoyl groupin position 3 activated hGHSR1a as efficiently as ghrelin, whilecompound 11 having a smaller benzoyl group in the same position was2-fold less potent.

[0119] Table 2 illustrates the effect of attaching the n-octanoyl groupto the side chain of residue 3 through an amide bond. In compounds 12and 14, 2,3-di-amino-propionic acid was incorporated in place of Ser³and the β-amino group of this new residue was acylated with n-octanoicacid. Compounds of 12 and 14 activated hGHSR1a as efficiently as theparent compounds, ghrelin and the compound 13 (peptide encompassing 1 to14 residues of ghrelin).

[0120] Table 3 provides binding and functional data on ghrelin analogswith the hydroxyl groups of serine residues other then Ser³ esterifiedby n-octanoic acid. Compound 15 with modified Ser², was active.Compounds 16 and 17, with modified Ser⁶ or Ser¹⁸, were inactive even atmicromolar concentrations.

[0121] Table 4 provides binding and functional data on truncatedanalogs. Shortening of ghrelin down to its 4 amino terminus residuesyielded active peptides. The relative binding affinities graduallydecreased with the extent of the C-terminal deletions. Short peptidesencompassing residue 1-3, or 2-5, or 3-5, or 2-4 of ghrelin were pooractivators even at micromolar concentrations. TABLE 1 Analogs of HumanGhrelin Modified at Position 3 Functional Assay % activation Binding at10 μM Assay relative to Compound X IC₅₀ (nM) EC₅₀ (nM) ghrelin HumanCO—(CH₂)₆CH₃ 0.25 ± 0.07  32 ± 4.5 100 Ghrelin 1CO—CH═CH—CH═CH—CH═CH—CH₃ 0.98 ± 0.36 39 ± 10 108 ± 1  2CO—CH(CH₂CH₂CH₃)₂ 0.96 ± 0.05 38 ± 11 103 ± 1  3 CO—(CH₂)₉CH₃ 0.12 ±0.03 9.1 ± 6.2 104 ± 3  4 CO—(CH₂)₁₄CH₃ 0.87 ± 0.17 8.3 ± 0.6  96 ± 11 5CO—CH₃   >2000 2000 ± 480   59 ± 13 6 — >10,000 >10,000 41 ± 4 7CO—(CH₂)₆CH₂Br 0.08 ± 0.0   18 ± 0.9 88 ± 7 8 CO—(CH₂)₂CO—NH—(CH₂)₂CH₃1020 ± 202  410 ± 120  86 ± 10 9 CO—(CH₂)₆NH₂   >2000 1200 ± 370  68 ± 310

0.12 ± 0.05  24 ± 9.5 95 ± 7 11

 11 ± 1.5  53 ± 3.2 85 ± 2

[0122] The modification shown in Table 1 is with respect to:   X   |GSSFLSPEHQRVQQRKESKKPPAKLQPR 1                         28

[0123] Compounds 1-11 correspond to SEQ. ID. NOs. 7-17. TABLE 2 Analogsof Human Ghrelin with an Amide Bond in the Side Chain of Residue 3Functional Assay Binding Assay % activation at 10 μM Compound IC₅₀ (nM)EC₅₀ (nM) relative to ghrelin Human Ghrelin 0.25 ± 0.07 32 ± 4.5 100 120.42 ± 12   31 ± 9.8 105 ± 5 13 9.6 ± 1.5 17 ± 4    97 ± 9 14   8 ± 2.738 ± 1.8 102 ± 3

[0124] Compound 12 is a modified amino acid having the followingsequence GSX^(b)FLSPEHQRVQQRKESKKPPAKLQPR (SEQ. ID. NO. 18), where X^(b)is

TABLE 3 Analogs of Human Ghrelin Octanoylated at Other Serine ResiduesFunctional Assay Binding Assay % activation at 10 μM Compound IC₅₀ (nM)EC₅₀ (nM) relative to ghrelin Human 0.25 ± 0.07 32 ± 4.5 100 Ghrelin 15 48 ± 7.2 42 ± 14   81 ± 14 16 >1000 >10,000 36 ± 1 17 >5000 >10,000 46± 2

[0125] Compounds 15-17 are as follows: (compound 15, SEQ. ID. NO. 19)GXSFLSPEHQRVQQRKESKKPPAKLQPR, (compound 16, SEQ. ID. NO. 20)GSSFLXPEHQRVQQRKESKKPPAKLQPR, (compound 17, SEQ. ID. NO. 21)GSSFLSPEHQRVQQRKEXKKPPAKLQPR,

[0126] wherein X is TABLE 4

Truncate Analogs of Human Ghrelin Functional Assay Binding Assay %activation at 10 μM Compound IC₅₀ (nM) EC₅₀ (nM) relative to ghrelinHuman 0.25 ± 0.07  32 ± 4.5 100 Ghrelin 18 0.16 ± 0.02  15 ± 4.5 100 ±4  19 0.77 ± 0.18 22 ± 16  92 ± 16 13 9.6 ± 1.5 17 ± 4  97 ± 9 20 7.1 ±5.7 20 ± 6   89 ± 17 21 55 ± 10 11.5 ± 2.3  96 ± 7 22 889 ± 72  72 ± 2991 ± 4 23   >2000 1150 ± 120  30 ± 5 24 >10,000 >10,000 28 ± 125 >10,000 2500 ± 1200 29 ± 7 26 >10,000 >10,000 28 ± 1

[0127] Compounds 23-26 are as follows:

[0128] Ac-SXFL-NH₂ (compound 23),

[0129] GSX-NH₂ (compound 24),

[0130] Ac-XFL-NH₂ (compound 25),

[0131] Ac-SXF-NH₂ (compound 26),

[0132] where X is

[0133] Other embodiments are within the following claims. While severalembodiments have been shown and described, various modifications may bemade without departing from the spirit and scope of the presentinvention.

1 22 1 28 PRT Human Xaa = Ser(CO-(CH2)6-CH3) 1 Gly Ser Xaa Phe Leu SerPro Glu His Gln Arg Val Gln Gln Arg Lys 1 5 10 15 Glu Ser Lys Lys ProPro Ala Lys Leu Gln Pro Arg 20 25 2 14 PRT Artificial Sequence Ghrelinanalog 2 Gly Ser Xaa Phe Leu Ser Pro Glu His Gln Arg Val Gln Gln 1 5 103 14 PRT Artificial Sequence Ghrelin analog 3 Gly Ser Xaa Phe Leu SerPro Glu His Gln Arg Val Gln Gln 1 5 10 4 23 PRT Artificial SequenceGhrelin analog 4 Gly Ser Xaa Phe Leu Ser Pro Glu His Gln Arg Val Gln GlnArg Lys 1 5 10 15 Glu Ser Lys Lys Pro Pro Ala 20 5 18 PRT ArtificialSequence Ghrelin analog 5 Gly Ser Xaa Phe Leu Ser Pro Glu His Gln ArgVal Gln Gln Arg Lys 1 5 10 15 Glu Ser 6 10 PRT Artificial SequenceGhrelin analog 6 Gly Ser Xaa Phe Leu Ser Pro Glu His Gln 1 5 10 7 28 PRTArtificial Sequence Ghrelin analog 7 Gly Ser Xaa Phe Leu Ser Pro Glu HisGln Arg Val Gln Gln Arg Lys 1 5 10 15 Glu Ser Lys Lys Pro Pro Ala LysLeu Gln Pro Arg 20 25 8 28 PRT Artificial Sequence Ghrelin analog 8 GlySer Xaa Phe Leu Ser Pro Glu His Gln Arg Val Gln Gln Arg Lys 1 5 10 15Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg 20 25 9 28 PRTArtificial Sequence Ghrelin analog 9 Gly Ser Xaa Phe Leu Ser Pro Glu HisGln Arg Val Gln Gln Arg Lys 1 5 10 15 Glu Ser Lys Lys Pro Pro Ala LysLeu Gln Pro Arg 20 25 10 28 PRT Artificial Sequence Ghrelin analog 10Gly Ser Xaa Phe Leu Ser Pro Glu His Gln Arg Val Gln Gln Arg Lys 1 5 1015 Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg 20 25 11 28 PRTArtificial Sequence Ghrelin analog 11 Gly Ser Xaa Phe Leu Ser Pro GluHis Gln Arg Val Gln Gln Arg Lys 1 5 10 15 Glu Ser Lys Lys Pro Pro AlaLys Leu Gln Pro Arg 20 25 12 28 PRT Artificial Sequence Ghrelin analog12 Gly Ser Ser Phe Leu Ser Pro Glu His Gln Arg Val Gln Gln Arg Lys 1 510 15 Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg 20 25 13 28 PRTArtificial Sequence Ghrelin analog 13 Gly Ser Xaa Phe Leu Ser Pro GluHis Gln Arg Val Gln Gln Arg Lys 1 5 10 15 Glu Ser Lys Lys Pro Pro AlaLys Leu Gln Pro Arg 20 25 14 28 PRT Artificial Sequence Ghrelin analog14 Gly Ser Xaa Phe Leu Ser Pro Glu His Gln Arg Val Gln Gln Arg Lys 1 510 15 Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg 20 25 15 28 PRTArtificial Sequence Ghrelin analog 15 Gly Ser Xaa Phe Leu Ser Pro GluHis Gln Arg Val Gln Gln Arg Lys 1 5 10 15 Glu Ser Lys Lys Pro Pro AlaLys Leu Gln Pro Arg 20 25 16 28 PRT Artificial Sequence Ghrelin analog16 Gly Ser Xaa Phe Leu Ser Pro Glu His Gln Arg Val Gln Gln Arg Lys 1 510 15 Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg 20 25 17 28 PRTArtificial Sequence Ghrelin analog 17 Gly Ser Xaa Phe Leu Ser Pro GluHis Gln Arg Val Gln Gln Arg Lys 1 5 10 15 Glu Ser Lys Lys Pro Pro AlaLys Leu Gln Pro Arg 20 25 18 28 PRT Artificial Sequence Ghrelin analog18 Gly Ser Xaa Phe Leu Ser Pro Glu His Gln Arg Val Gln Gln Arg Lys 1 510 15 Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg 20 25 19 28 PRTArtificial Sequence Ghrelin analog 19 Gly Xaa Ser Phe Leu Ser Pro GluHis Gln Arg Val Gln Gln Arg Lys 1 5 10 15 Glu Ser Lys Lys Pro Pro AlaLys Leu Gln Pro Arg 20 25 20 28 PRT Artificial Sequence Ghrelin analog20 Gly Ser Ser Phe Leu Xaa Pro Glu His Gln Arg Val Gln Gln Arg Lys 1 510 15 Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg 20 25 21 28 PRTArtificial Sequence Ghrelin analog 21 Gly Ser Ser Phe Leu Ser Pro GluHis Gln Arg Val Gln Gln Arg Lys 1 5 10 15 Glu Xaa Lys Lys Pro Pro AlaLys Leu Gln Pro Arg 20 25 22 1101 DNA Artificial Sequence Human GHSreceptor cDNA 22 atgtggaacg cgacgcccag cgaagagccg gggttcaacc tcacactggccgacctggac 60 tgggatgctt cccccggcaa cgactcgctg ggcgacgagc tgctgcagctcttccccgcg 120 ccgctgctgg cgggcgtcac agccacctgc gtggcactct tcgtggtgggtatcgctggc 180 aacctgctca ccatgctggt ggtgtcgcgc ttccgcgagc tgcgcaccaccaccaacctc 240 tacctgtcca gcatggcctt ctccgatctg ctcatcttcc tctgcatgcccctggacctc 300 gttcgcctct ggcagtaccg gccctggaac ttcggcgacc tcctctgcaaactcttccaa 360 ttcgtcagtg agagctgcac ctacgccacg gtgctcacca tcacagcgctgagcgtcgag 420 cgctacttcg ccatctgctt cccactccgg gccaaggtgg tggtcaccaaggggcgggtg 480 aagctggtca tcttcgtcat ctgggccgtg gccttctgca gcgccgggcccatcttcgtg 540 ctagtcgggg tggagcacga gaacggcacc gacccttggg acaccaacgagtgccgcccc 600 accgagtttg cggtgcgctc tggactgctc acggtcatgg tgtgggtgtccagcatcttc 660 ttcttccttc ctgtcttctg tctcacggtc ctctacagtc tcatcggcaggaagctgtgg 720 cggaggaggc gcggcgatgc tgtcgtgggt gcctcgctca gggaccagaaccacaagcaa 780 accgtgaaaa tgctggctgt agtggtgttt gccttcatcc tctgctggctccccttccac 840 gtagggcgat atttattttc caaatccttt gagcctggct ccttggagattgctcagatc 900 agccagtact gcaacctcgt gtcctttgtc ctcttctacc tcagtgctgccatcaacccc 960 attctgtaca acatcatgtc caagaagtac cgggtggcag tgttcagacttctgggattc 1020 gaacccttct cccagagaaa gctctccact ctgaaagatg aaagttctcgggcctggaca 1080 gaatctagta ttaatacatg a 1101

What is claimed is:
 1. A truncated ghrelin analog having a structureselected from the group consisting of: a) Z¹-GSXF(Z)_(n)-Z²; and b)Z¹-GXSF(Z)_(n)-Z^(2;) wherein X is a modified amino acid containing abulky hydrophobic R group; each Z is independently either alanine,valine, leucine, isoleucine, proline, tryptophan, phenylalanine,methionine, glycine, serine, threonine, tyrosine, cysteine, asparagine,glutamine, lysine, arginine, histidine, aspartic acid, or glutamic acid,or a derivative thereof; Z¹ is an optionally present protecting groupthat, if present, is covalently joined to the N-terminal amino group; Z²is an optionally present protecting group that, if present, iscovalently joined to the C-terminal carboxy group; and n is 0 to 19; ora pharmaceutically acceptable salt thereof.
 2. The analog of claim 1,wherein X has the structure:

wherein X¹ is either —O—, —S—, —OC(O)—, —NHC(O)—, or —CH₂—; and R iseither —C₄₋₂₀ alkyl, —C₄₋₂₀ substituted alkyl, —C₄₋₂₀ substitutedalkenyl, —C₄₋₂₀ alkenyl, —C₄₋₂₀ heteroalkyl, —C₄₋₂₀ substitutedheteroalkyl, aryl, or alkylaryl.
 3. The analog of claim 2, wherein saidanalog has the structure: Z¹-GSXF(Z)_(n)-Z².
 4. The analog of claim 3,wherein n is 0-11.
 5. The analog of claim 4, wherein n is 0-6.
 6. Theanalog of claim 5, wherein n is 0-3.
 7. The analog of claim 6, wherein nis
 0. 8. The analog of any one of claims 1-7, wherein X¹ is —C(O)— or—NH(O)—; and R is a —C₅₋₁₅ alkyl.
 9. The analog of claim 8, wherein X¹is —C(O)— and R is —(CH₂)₆CH₃.
 10. The analog of claim 9, wherein Z¹ ifpresent is —C(O)CH₃ and Z² if present is —NH₂.
 11. The analog of claim1, wherein said analog has a modified amino acid sequence selected fromthe group consisting of: GSXFLSPEHQRVQQRKESKKPPA-NH₂ (SEQ. ID. NO. 4),GSXFLSPEHQRVQQRKES-NH₂ (SEQ. ID. NO. 5), GSXFLSPEHQRVQQ (SEQ. ID. NO.2), GSX^(b)FLSPEHQRVQQ (SEQ. ID. NO. 3), GSXMLSPEHQ-NH₂ (SEQ. ID. NO.6), GSXFL-NH₂ and GSXF-NH₂; provided that X is

or a pharmaceutically acceptable salt thereof.
 12. A method of screeningfor a compound able to bind to a growth-hormone secretagogue receptorcomprising the step of measuring the ability of said compound to effectbinding of the analog of claim 1 to either said receptor, a fragment ofsaid receptor comprising a ghrelin binding site, a polypeptidecomprising said fragment, or a derivative of said polypeptide.
 13. Themethod of claim 12, wherein said method measures the ability of saidanalog to bind to said receptor.
 14. The method of claim 13, whereinsaid analog is selected from the group consisting of:GSXFLSPEHQRVQQRKESKKPPA-NH₂ (SEQ. ID. NO. 4), GSXFLSPEHQRVQQRKES-NH₂(SEQ. ID. NO. 5), GSXFLSPEHQRVQQ (SEQ. ID. NO. 2), GSX^(b)FLSPEHQRVQQ(SEQ. ID. NO. 3), GSXFLSPEHQ-NH₂ (SEQ. ID. NO. 6), GSXFL-NH₂ andGSXF-NH₂; provided that X is

or a pharmaceutically acceptable salt thereof.
 15. A method forachieving a beneficial effect in a subject comprising the step ofadministering to the subject an effective amount of the analog ofclaim
 1. 16. The method of claim 15, wherein said analog is selectedfrom the group consisting of: GSXFLSPEHQRVQQRKESKKPPA-NH₂ (SEQ. ID. NO.4), GSXFLSPEHQRVQQRKES-NH₂ (SEQ. ID. NO. 5), GSXFLSPEHQRVQQ (SEQ. ID.NO. 2), GSX^(b)FLSPEHQRVQQ (SEQ. ID. NO. 3), GSXFLSPEHQ-NH₂ (SEQ. ID.NO. 6), GSXFL-NH₂ and GSXF-NH₂; provided that X is

or a pharmaceutically acceptable salt thereof.
 17. A method forstimulating growth hormone secretion comprising the step ofadministering to a subject an effective amount of the analog of claim 1.18. The method of claim 17, wherein said analog is selected from thegroup consisting of: GSXFLSPEHQRVQQRKESKKPPA-NH₂ (SEQ. ID. NO. 4),GSXFLSPEHQRVQQRKES-NH₂ (SEQ. ID. NO. 5), GSXFLSPEHQRVQQ (SEQ. ID. NO.2), GSX^(b)FLSPEHQRVQQ (SEQ. ID. NO. 3), GSXFLSPEHQ-NH₂ (SEQ. ID. NO.6), GSXFL-NH₂ and GSXF-NH₂; provided that X is

or a pharmaceutically acceptable salt thereof.